The determination of heparin establishes an important parameter for the supervision of heparin treatment which is often administered in the presence of a threat of thrombosis. Heparin forms with antithrombin III (AT III) a complex which inhibits the proteolytic activity of thrombin. Since thrombin catalyzes the formation of fibrin from fibrinogen, the thrombin activity is responsible for the coagulation of blood or plasma and hence also for the fibrin clots which form in thrombosis. Heparin treatment is often applied in the presence of a threat of thrombosis (e.g. before surgical interventions). A precise adjustment of heparin concentration is therefore extremely important. If the dose is too low, there is the danger of thrombosis or embolism, which can result in death. Excessively high heparin concentrations, however, result in bleeding. The quantitative analysis of heparin, therefore, is one of the tests most frequently performed in the blood testing laboratory.
In 1973 Yin and co-workers developed the first quantitative assay method for the in vitro measurement of heparin in plasma based on Factor X.sub.a neutralization (J. Lab. Clin. Med. 81: 298, 1973). This assay method had the advantage over the methods known prior to 1973 in that it could detect less than 0.02 units of heparin per milliliter of plasma. However, the assay is cumbersome and time consuming to perform. It is a two-stage assay, and requires much manual manipulation. In the first-stage of the assay, the patient's plasma sample is incubated with normal plasma, buffer, and a known excess of Factor X.sub.a for a predetermined time period, after which a sub sample from this primary reaction mixture is removed and assayed for Factor X.sub.a activity. The latter step constitutes the second-stage of the assay. Factor X.sub.a activity is measured by the addition of the test sample to another test tube, and to it calcium chloride, cephalin in bovine plasma are added separately in timed fashion. A commercial embodiment of this method is described in detail in Sigma Chemical Company Technical Bulletin No. 870 (as revised in Jan. 1982). A copy of this bulletin is attached hereto and its disclosure is incorporated by reference.